The calcium dependent proteases known as calpains contribute significantly to neuronal degeneration in chronic diseases, such as Alzheimer's, Huntington's or Parkinson's; and in response to acute injury including trauma or stroke. In contrast to the evidence suggesting adverse roles for calpain(s) in pathologic conditions, proper calpain function is required for embryonic development and fundamental processes involving motility such as cellular migration including neuronal growth cone motility and guidance. Therefore it is important to fully understand when and where calpain functions in both health and disease/injury states. We currently lack the ability to selectively visualize activated calpain within cells; i.e. we lack methodology that allows spatio-temporal and dynamic resolution of functional calpain. The first aim of this proposal is to develop a FRET generating method to selectively identify active, Ca2+ bound, calpain. Calpastatin, the specific endogenous inhibitor protein of calpain, and its well characterized interaction with Ca2+-calpain provide the framework for design of the sensor. Two alternative approaches are proposed based on the 1) proximity of the bound inhibitor to the C-terminus of calpain; 2) the proximity of the two termini of bound calpastatin to one another; and 3) the independence of the binding interactions between each calpastatin subdomain ("A", "B", "C") and calpain. Optimization of the sensor design will involve in vitro validation of the Ca2+-dependence of FRET and characterization of the interaction between the probe with the ubiquitous calpains-1 and 2. Future extension of this technology is expected to aid development of calpain specific substrates; with potential for isoform specific substrate development. The second aim will assess the utility of the sensor in situ using cultured Xenopus spinal neurons and cultured fibroblastic (Chinese hamster ovary) cells. Each of these models has been previously characterized with regard to the calpain system and its responsiveness to defined conditions. Results of imaging activation of calpain directly via the FRET generating probe will be compared with prior results based on the indirect, currently available, methods. [unreadable] [unreadable]